Ionizing radiation, mutagenic chemicals, and replication of a damaged DNA template create DNA double-stranded breaks. If not handled properly, these breaks can lead to gross chromosome rearrangements and genome destabilization. Homologous recombination represents a major, conserved mechanism for the elimination of DNA double-strand breaks. Importantly, there is compelling evidence showing that even a minor deficiency in HR leads to disorders with a strong cancer predisposition in humans. Understanding the mechanisms of DSB repair by HR is highly relevant to radiation biology and also cancer biology. Over the past several decades, there has been great progress in identifying key enzymes and dissecting the mechanisms of HR, with major contributions coming from studies in the budding yeast S. cerevisiae. In this renewal project, we will tackle several outstanding questions germane for delineating the multi-faceted roles of the conserved DNA helicases Pif1 and Mph1 in the DNA synthesis step of recombination and recombination pathway choice, respectively. Moreover, we will determine how the Smc5-Smc6 complex influences the activity of the Mph1 helicase in DNA replication fork repair. The results from our continuing efforts will advance our understanding of the formation and resolution of DNA intermediates during the homologous repair of damaged chromosomes.